methylation analysis - methylation analysis using epitect bisulfite kit from qiagen (Jan/15/2008 )
Hi davo,
sry for my late answer, but i´m using the Qiagen Hot Start. For other sequences the
Peq Lab Hot Start works also fine.
For Longer fragments you could try the KOD Hot Start from Merck.
I know this is a lot of work but once you choose the right Polymerase it will work better.
Also one more tip: I add Betaine and TMAC to my PCR´s , this is also a great advantage. Specially
Betaine is supposed to reduce the BIAS in the PCR.
Good look
-Dani_Bisulfite-
QUOTE (ghai @ Jan 15 2008, 06:55 PM)
hi every body there
is there any body doing methylation analysis using epitect bisulfite kit from qiagen
actually I am facing the problem that after bisulfite treatment and purification, when i load the sample in 1.5% agarose no band is visible.
I started with 2ug of DNA and eluted in 30ul of EB buffer and loaded 6ul on agarose gel
can anybody help me to troubleshoot the problem??
Sandeep Ghai
is there any body doing methylation analysis using epitect bisulfite kit from qiagen
actually I am facing the problem that after bisulfite treatment and purification, when i load the sample in 1.5% agarose no band is visible.
I started with 2ug of DNA and eluted in 30ul of EB buffer and loaded 6ul on agarose gel
can anybody help me to troubleshoot the problem??
Sandeep Ghai
Hi Ghai,
I had the same problem also: no bisulfite DNA coming of your column. I use another kit, but the problem looks similar.
What I noticed, after analysing yields etc. etc. after doing many many bisulfite treatments, that the amount of bisulfite DNA coming of the column drops when the genomic DNA is contaminated with either RNA or protein (low or to high 260/280 and 260/230). And it also is shitty if your gDNA is degraded. So check the integrety of your gDNA (0.7% agarose gel, one nice big bad bloba high on the gel and no larger smear) and check the purity (260/280 1.75-1.85). Hope it helps
Happy bisulfite treatments!
ET2B
-ET2B-